The activity of apoptosis protein BNIP3 has been associated with its ability to form homodimeric and heteromeric associations through its carboxy-terminal transmembrane domain (TMD), but little is known about the chemical or physical basis of these interactions. In this thesis, I describe two approaches to examine the sequence requirements for BNIP3 TMD dimerization and the properties that drive and stabilize this association. The first approach employs saturation mutagenesis to generate a library of single mutants in the context of a fusion protein construct and SDS-PAGE combined with Western blotting to characterize the mutant dimerization phenotypes. The mutagenesis data maps the BNIP3 TMD dimerization region and identifies five interacting residues for BNIP3 TMD dimerization: Ala176, Gly180, and Gly184 form tandem GxxxG motifs that allow close approach of the helix backbones, and His173 and Ser172 form inter-monomer hydrogen bonds. The mutagenesis data also show that the sequence context in which these five critical residues are embedded affects the strength of TMD helix-helix interactions because several mutations at or near the dimer interface that leave the small residues and the inter-monomer hydrogen bonding intact can abolish or profoundly lower dimerization. The second approach to the study of BNIP3 TMD dimerization involves determining the structure of the TMD dimer using solution NMR spectroscopy. Reconstitution of the BNIP3 TMD peptide in the non-ionic detergent dodecylphosphocholine (DPC) and addition of dipalmitoylphosphatidylcholine (DPPC) yields peptide spectra with excellent peak dispersion and resolution. This quality enables collection of chemical shift, J coupling, and NOE distance restraints, thus allowing determination of the BNIP3 TMD dimer structure. The NMR structure of the BNIP3 TMD dimer reveals the details of how the elements of the BNIP3 TMD sequence cooperate to support dimerization and provides a context to interpret the effects of the saturation mutagenesis results. Results from the saturation mutagenesis and structural analyses establish an understanding of the BNIP3 TMD dimerization and provide a framework for further studies of BNIP3, which include but are not limited to thermodynamic studies, functional analyses, and molecular dynamics modeling of the BNIP3 TMD associations.

Applications of recombinant DNA technology to protein analysis have been demonstrated using the trimethoprim resistant (Tp$\sp{\rm R}$)R388 type II dihydrofolate reductase (DHFR). DHFR fusion vectors were constructed which contain cloning sites in the C-terminal coding region of the DHFR. Segments of nematode major sperm protein (MSP) fused to the DHFR were recognized by antibody to MSP. A 15 aa segment of MSP sequence, fused to the DHFR, was shown to be sufficient to elicit antibody to MSP. A two-step procedure was developed for purifying the DHFR proteins. A selectable FokI/supF cassette was developed to facilitate DNA excision and replacement mutagenesis. When the cassette is inserted into DNA, the presence of the tyrosine tRNA suppressor gene (supF) contained on the cassette is selected by the suppression of amber mutations in the recipient host. Subsequent refined mutagenesis is possible due to the unique cleavage properties of FokI. The cassette/vector system was used to produce a deletion corresponding to amino acid residues 2-7 of the DHFR which did not noticeably impair Tp$\sp{\rm R}$. Resistance was abolished, however, by a deletion of amino acid residues 2-21. A structural gene was synthesized which contains many unique restriction sites and encodes a Tp$\sp{\rm R}$ DHFR which is 10 aa shorter at the N-terminus relative to natural type II DHFRs. High copy number vectors which utilize strong promoters to transcribe the dhfr gene and the primer for plasmid replication were constructed to overproduce the mini DHFR and a full-length derivative. Site-directed mutagenesis was used to test the importance of glu-58 and thr-48 in a putative folate binding site of the mini and full-length DHFRs. The introduction of thr-58 and/or glu-48 destroyed in vivo function of the DHFRs. Tp$\sp{\rm R}$ was retained in the full length gln-58 R388 DHFR, but not in the mini gln-58 DHFR. Through random mutagenesis a mini Tp$\sp{\rm R}$gln-58 R388 DHFR was obtained that contained a duplication of leu-pro-ser, at the N-terminus. Successive additions of the leu-pro-ser triplet to the N-terminus appear to stabilize the functional form of the mini DHFR.

3$\beta$-Hydroxy-5$\alpha$-cholest-8(14)-en-15-one (15-ketosterol) is a hypocholesterolemic agent which affects cholesterol metabolism at several levels, including sterol biosynthesis and cholesterol absorption. This dissertation describes studies on the effect of 15-ketosterol on intestinal acyl CoA:cholesterol acyltransferase (ACAT) activity, an enzyme involved in the esterification and absorption of dietary cholesterol. Addition of 15-ketosterol to jejunal microsomes decreased the level of cholesterol esterified by ACAT. Incubation of 15-ketosterol with rat jejunal microsomes reduced the (1 -$\sp{14}$C) oleoyl CoA-dependent esterification of microsomal cholesterol (50% inhibition: 3.0 $\mu$M). This reduction in cholesterol esterification was accompanied by the formation of ($\sp{14}$C) labeled material which, upon analysis by thin layer chromatography, comigrated with 15-ketosteryl oleate. The esterification of 15-ketosterol was confirmed by incubating (2,4- 3H) 15-ketosterol with unlabeled oleoyl CoA and jejunal microsomes. Analyses using either normal phase thin layer chromatography or reverse phase high pressure liquid chromatography detected the formation of (3H) material comigrating with 15-ketosteryl oleate. Oral administration of 15-ketosterol to rats lowered ACAT activity relative to pair-fed controls. ACAT activity in rat jejunal microsomes was lowered 82% (P $<$ 0.02) and 77% (p $<$ 0.001) in animals fed a chow diet containing 0.05% and 0.10% 15-ketosterol, respectively. Analysis of the cholesterol and cholesterol ester content of rat jejunal microsomes showed that oral administration of either 0.10% or 0.125% 15-ketosterol did not affect the concentrations of these compounds. Additional studies showed that reduction of rat jejunal ACAT activity was dependent upon both the duration of oral administration of 15-ketosterol and the concentration of the compound in the diet. Reduction of ACAT activity was observed as early as 3 hours after ingestion of the first meal containing 0.10% 15-ketosterol. Significant reductions in microsomal ACAT activity were observed at concentrations as low as 0.05% 15-ketosterol in the diet. Little or no incorporation of (1-$\sp{14}$C) oleoyl CoA into material comigrating with 15-ketosteryl oleate was observed. In additional studies, incubation of 10 $\mu$M (25R)-3$\beta$,26-dihydroxy-5$\alpha$-cholest-8(14)-en-15-one, a metabolite of 15-ketosterol, with rat jejunal microsomes resulted in a 55% reduction in the oleoyl CoA-dependent esterification of microsomal cholesterol.

CO-heme interactions with sperm whale apomyoglobin (apoMb) and model membrane systems were examined by spectroscopic and kinetic techniques. The binding of heme to apoMb appears to be a simple bimolecular process at relatively low apoprotein concentrations. The association rate constant, 3 $\times$ 10$\sp7$ M$\sp{-1}$s$\sp{-1}$, was measured directly at 10$\sp\circ$C. The circular dichroism spectra for newly reconstituted Mb was initially decreased when compared to native Mb. The half time for equilibration to the native state can range from several hours to days depending on the pH, ligand, and oxidation state. The rate constants for the association or dissociation of oxygen or carbon monoxide to newly formed Mb were identical to native Mb. Kinetic heterogeneity was observed in long-chain isonitrile binding. These results suggest that although rapid uptake of heme by the apoprotein can result in two orientations, there are no apparent changes in the physiological properties of myoglobin. The effects of vesicle composition on heme binding to membranes were probed using thirty separate lipid mixtures. A sharp decrease in the rate of heme binding to liposomes was observed as the lipid vesicles changed form liquid-crystalline to gel phase. The addition of dicetyl phosphate, which has a negative charge at neutral pH, generally decreased the overall rate and affinity of the vesicles for heme binding. The rate and extent of heme uptake in unsaturated lecithins is unaffected by cholesterol content at levels up to 40% per mole. In contrast, the affinity of saturated dimyristoylphosphatidylcholine (DMPC) vesicles for heme dramatically decreased with increasing cholesterol content. This effect appears to be related to the influence of cholesterol on the DMPC phase transition temperature ($T\sb{m}$). Although there is some variation, most lecithins with low $T\sb{m}$ values have an overall equilibrium partition constant $\approx$5 $\times$ 10$\sp5$ and association and dissociation rate constants $\approx$3 $\times$ 10$\sp6$ s$\sp{-1}$ and 7 s$\sp{-1}$, respectively, at 30$\sp\circ$C. Association, dissociation, column chromatography, and temperature dependence studies of CO-heme binding to liposomes indicate that the rate of heme transmembrane movement is slow and dependent on phosphatidylcholine acyl-chain length. The rate increases sharply at the phase transition temperature.

The physiological role of myoglobin depends on the modulation of heme activity by the protein. The hypothesis that functional properties are governed by conserved residues in the distal pocket has been tested by site-directed mutagenesis of three residues: Leu$\sp{29}$, His$\sp{64}$, and Val$\sp{68}$. To facilitate interpretation of functional data, structures of several mutant myoglobins have been determined by X-ray crystallography. Leu$\sp{29}$ controls the volume of the distal pocket. Since Val$\sp{29}$ does not contact bound ligands, this substitution does not affect ligand affinities significantly. It does permit solvent approach to the heme, thereby increasing the rate of autooxidation. Although the Phe$\sp{29}$ mutant was constructed to reduce the volume of the binding site, dipole-multipole interactions stabilize bound oxygen and reduce the rate of autooxidation substantially. His$\sp{64}$ inhibits oxygen dissociation and autooxidation by hydrogen bonding to the ligand. In conjunction with a distal water molecule, it sterically hinders carbon monoxide association. Mutation of this residue eliminates hydrogen-bonding interactions in all cases except Gln$\sp{64}$, producing low oxygen affinities and high rates of autooxidation. In the Gly$\sp{64}$ mutant, the solvent-filled distal cavity partially restores binding site polarity. In contrast, the distal pockets of Val$\sp{64}$, Thr$\sp{64}$, and Leu$\sp{64}$ are completely apolar, leading to marked increases in rates of ligand binding. Val$\sp{68}$ governs the ligand accessibility of the iron. In the Ala$\sp{68}$ mutant, only slight rate enhancements occur because the distal water molecule is retained in the deoxygenated protein. The larger side chains of Ile$\sp{68}$ and Leu$\sp{68}$ displace this water molecule and occlude the binding site in unliganded structures. The lower affinities observed in Ile$\sp{68}$ compared to Leu$\sp{68}$ are due to the decreased ability of this residue to accommodate the bound ligand. In contrast, the Phe$\sp{68}$ side chain is directed away from the iron atom and does not inhibit binding directly. Nevertheless, the reduced volume in this mutant is filled with a water molecule, retarding ligand association. In all mutants, structural perturbations are limited to the site of the substitution and the flexible corner regions of myoglobin. Furthermore, the stereochemistry of the heme-ligand complex is little influenced by changes in the distal pocket.

This research investigated the effects of temperature, pH, and anaerobiosis on gene expression in Escherichia coli. In the first series of experiments, the effects of FirA, a histone-like protein, and two temperature sensitive mutants on chromosomal lac operon expression were examined. The mutant Fir proteins have no effect on the transcription of the lac operon at 30$\sp\circ$C or upon a temperature shift to 40$\sp\circ$C. The effects of temperature on in vivo expression of E. coli promoters were also examined. The tet and anti-tet promoters of pBR322, lacUV5, trp, several hybrid promoters, and spacing variants of the tet promoter followed the general trend of increasing expression level between 20$\sp\circ$C and 37$\sp\circ$C with a leveling or some decrease in expression above 37$\sp\circ$C. Only the tet::trp promoter and the consensus sequence synthetic tet promoters deviated from this pattern, increasing in activity above 40$\sp\circ$C. No effects of temperature were observed on the expression profiles of trp promoters with altered flanking sequences. The effects of temperature on the induction of the trp promoter by 3-$\beta$-indole acrylic acid were examined. Five $\mu$g/ml was sufficient to derepress the trp promoter at the same induction ratio from 20$\sp\circ$C to 45$\sp\circ$C. The biodegradative lysine decarboxylase (LDC) of E. coli is induced by low pH, anaerobiosis, and excess lysine. A series of isogenic E. coli strains were constructed to determine the effects of CadR, Fnr, Cya, CRP, and Pgi on LDC expression. These strains were grown in a rich medium similar to Falkow decarboxylase medium at pH 5.5, pH 6.8, and pH 8.0 under aerobic and anaerobic conditions. The pH and air responses were retained in these mutants demonstrating that these regulatory processes do not mediate LDC regulation. To further study the regulation of biodegradative LDC, as well as biodegradative arginine decarboxylase, a Mu lac fusion library was constructed. Stable LDC deficient lac fusions which are regulated by low pH or anaerobic conditions were identified. Two stable arginine decarboxylase deficient lac fusions were observed to be regulated by low pH and anaerobic conditions. Three LDC deficient lac fusion strains regulated by low pH or anaerobiosis have been mapped to the cadA gene.

Clones containing DNA uniquely transcribed during oogenesis were isolated from genomic DNA phage libraries for the two related Dipteran flies C. erythrocephala and D. melanogaster. Poly(A)$\sp{+}$ RNA from early or mid-stage ovarian follicles of C. erythrocephala was used to generate radiolabelled oogenesis-specific cDNA probes for screening the phage libraries. A cDNA probe made from mid-stage embryo poly(A)$\sp{+}$ RNA was used as the differential screening probe. Thus plaques hybridizing to the two oogenesis-specific probes but not the mid-stage embryo probe were selected as potentially containing oogenesis-specific genes. Two further rounds of screening were used to eliminate false positives and, after plaque purification, restriction digests of the remaining clones were screened by Southern blot hybridization to identify DNA fragments transcribed in an oogenesis-specific manner. To date, 22 oogenesis-specific clones have been isolated from C. erythrocephala and two from D. melanogaster by this method. In situ hybridization to sections of ovarian follicles has been used to determine the cell types within the follicles in which the various genes are expressed. Radiolabelled RNA probes for four of the C. erythrocephala oogenesis-specific clones and the two D. melanogaster clones have been hybridized to ovarian follicles. Two of the C. erythrocephala clones and both of the D. melanogaster clones have thus been shown to be transcribed in the germ-like cells of the follicles as opposed to the somatic follicle cells. Further studies have been concentrated on the two germ-line transcribed, oogenesis-specific clones isolated form the D. melanogaster clone library. In situ hybridization of the two D. melanogaster clones to the salivary gland polytene chromosomes has established that one clone is derived from chromosome region 31B/D (clone DA) and the second clone from region 98E/F (clone DM). Region 31B/D is rich in female sterile mutations and detailed genetic mapping of the DA clone and of these mutations was performed (using deficiency chromosomes) to determine which mutations might represent the DA gene. cDNA clones have been isolated for the transcribed region of clone DA and have been used to further define the transcription unit from this region of the D. melanogaster genome.

The interactions of Escherichia coli trp aporepressor and its ligands, tryptophan and trp operator DNA, were examined. To avoid the interference of fluorescence from the ligand, a hydrophobicity-dependent fluorophore, 8-anilino-1-naphthalenesulfonate (ANS), was used to monitor tryptophan binding. The fluorescence decrease of ANS caused by tryptophan competitive displacement was utilized to measure the affinity of tryptophan and its aporepressor. The results analyzed by the method of Horovitz and Levitzki (1987) showed the equilibrium dissociation constant to be 3 $\times$ 10$\sp{-5}$ M. This value is similar to the results obtained from equilibrium dialysis and other spectrometric methods. The kinetic parameters were measured by monitoring the tryptophan fluorescence difference directly on stopped-flow fluorospectrometer. The dissociation rate constant of tryptophan from trp aporepressor is about 50 s$\sp{-1}$. The operator binding assay was performed by gel retardation electrophoresis developed by Carey (1987). The apparent equilibrium dissociation constants for trp repressor and 40 or 90 base pair (bp) operator-containing DNA are identical. A dissociation rate of 5 $\times$ 10$\sp{-2}$ s$\sp{-1}$ was obtained by the displacement experiment with unlabeled operator DNA. This result was consistent with the value from nitrocellulose filter binding assay (Klig et al., 1987). Three point mutations of trp aporepressor, each with a serine to cysteine at positions 67, 86 and 88, were constructed by oligonucleotide-directed site-specific mutagenesis. Taking the advantage of the similarities between hydroxyl and sulfhydryl groups, these substitutions were made to provide sites to introduce fluorescent probes into the mutant proteins. However, both tryptophan and operator DNA binding affinities for all three mutants were decreased. These results may reflect the complexity within this region which includes the amino acid residues in the tryptophan binding site, DNA recognition site and hydrophobic brace (Zhang et al., 1987). Results from ultracentrifugation, polarization of fluorescence and gel filtration indicate that the trp aporepressor may undergo a concentration dependent association. The in vivo role of this association is not clear. However, the possibility of DNA loop formation through a protein-protein interaction is consistent with observations on several other gene regulatory proteins.

The sequences that comprise a polyadenylation signal are varied. With the exception of the conserved hexanucleotide AAUAAA, other required sequence elements vary from site to site. This variability in sequence content may be indicative of different types or classes of polyadenylation signals. We have used an in vitro polyadenylation system to investigate the possibility that classes of poly(A) sites exist. Precursor RNAs from the SV40 late and adenovirus 2 L3 polyadenylation sites were examined for differences in: assembly into RNA-protein complexes; sequence requirements for complex assembly; and interactions with small nuclear ribonucleoproteins (snRNPs). Both SV40 late and L3 precursor RNA required an intact hexanucleotide and downstream sequence elements for complex formation. The stability of the complexes assembled using the two precursor RNAs was different. The L3 RNA complex was unstable in the presence of the anion poly(ACU); whereas the SV40 late complex or a chimeric L3/SV40 late complex were not. SV40 late and L3 precursor RNAs associated with the Sm protein determinant (common to U1, U2, U4/U6, U5 and U7 snRNPs) and a U1 snRNP-specific protein determinant early in the polyadenylation reaction. This association was reduced as the polyadenylation reaction progressed. Formation of the polyadenylation specific complexes was shown to require the small nuclear RNAs (snRNAs) U1, U2 and U4. The two polyadenylation precursor RNAs showed a different sensitivity to oligonucleotide directed RNase H cleavage of U RNAs. The SV40 late site was sensitive to cleavages of U1, U2 and U4 RNA. The L3 site showed sensitivity to only U4 cleavage. When the two polyadenylation signals were preceded by a functional intron with 5$\sp\prime$ and 3$\sp\prime$ splice sites, sensitivity to U RNA cleavages was altered. However even as chimeric polyadenylation splicing templates, the two sites exhibited different sensitivities to U4 cleavage in the region of U4 in which it hybridizes to U6 RNA. The observed differences in complex stability, and sensitivity to U RNA cleavage suggest that different classes of polyadenylation sites exist.

As the classical model for negative transcriptional control in prokaryotes and the subject of concentrated experimental attention, the lactose operon of Escherichia coli presents a well-defined system for studying genetic control through protein-DNA binding interactions. Binding of repressor at its cognate operator sequence within the regulatory region of the operon, while responsive to environmental conditions, efficiently inhibits transcription initiation by RNA polymerase. The high binding affinity and degree of specificity exhibited by this protein-DNA complex has encouraged investigation of the nature of the contacts formed. We have explored specific contacts between the lac repressor and operator using 5-bromodeoxyuridine-substituted DNA. Substitution of BrdU for single thymidine positions in a synthetic 40 bp operator provides an indirect means of probing the major groove of operator DNA for critical contacts between the repressors and the 5-methyl of individual thymidines. As a photoreactive species, BrdU provides substrate for ultraviolet irradiation. Upon irradiation, strand scission occurs at the BrdU residues. When bound, lac repressor protein provides protection against UV-induced breakage depending on the nature of the sites and type of interaction. We have confirmed thirteen unique sites of inducer-sensitive protection along the operator sequence (+1, 2, 3, 4, 6, 8, 13, 15, 16, 18, 19, 20, 21) using this method compared to per-substitution with BrdU (Ogata and Gilbert, 1977). The ability of these photosensitive DNAs to form short-range cross-links to bound protein has been used to determine the efficiency with which cross-linked protein-DNA complexes are generated at each individual site of BrdU substitution. Five sites of high efficiency cross-linking to the repressor protein have been identified (+3, 4, 14, 18, 19). Comparison of the UV protection results and the cross-linking data shows that these processes provide complementary tools for identifying and analyzing individual protein-DNA contacts. Using these same BrdU-substituted operator DNAs, we attempted to define individual protein-DNA interactions with respect to the specific amino acid(s) making contact at a selected site within the operator sequence. With the selection of the T$\sb{+3}$ site for our initial investigation, the cross-linked complex was formed and isolated. These polypeptide-DNA species were prepared for final analysis through a series of steps including proteolysis and anion-exchange HPLC. Protein sequence analysis on the purified peptide-operator complex identified a peptide spanning Val23 through Lys33. The data suggest His29 as the specifically crosslinked amino acid.